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J. Biol. Chem., Vol. 278, Issue 47, 46293-46306, November 21, 2003

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N-Glycan Structures of Pigeon IgG

A MAJOR SERUM GLYCOPROTEIN CONTAINING Gal1–4Gal TERMINI^*

Noriko Suzuki, Kay-Hooi Khoo, Chin-Mei Chen, Hao-Chia Chen¶, and Yuan C. Lee||

From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, and ^¶Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 3, 2003 , and in revised form, September 4, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We had shown previously that all major glycoproteins of pigeon egg white contain Gal1–4Gal epitopes (Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, 23221–23229). We now report that Gal1–4Gal-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by Western blot with Griffonia simplicifolia-I lectin (specific for terminal -Gal) and anti-P₁ (specific for Gal1–4Gal1–4GlcNAc1–) monoclonal antibody. One of the major glycoproteins from pigeon plasma was identified as IgG (also known as IgY), which has Gal1–4Gal in its heavy chains. High pressure liquid chromatography, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included (i) high mannose-type (33.3%), (ii) disialylated biantennary complex-type (19.2%), and (iii) -galactosylated complex-type N-glycans (47.5%). Bi- and tri-antennary oligosaccharides with bisecting GlcNAc and 1–6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Gal1–4Gal sequences. Moreover, MS/MS analysis identified Gal1–4Gal1–4Gal1–4GlcNAc branch terminals, which are not found in pigeon egg white glycoproteins. An additional interesting aspect is that about two-thirds of high mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high mannose-type oligosaccharides may be a characteristic of these avian IgG.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Pigeon (Columba livia) egg white (PEW)¹ is a rich source of glycoproteins containing Gal1–4Gal (galabiose). All four major PEW glycoproteins, ovotransferrin, two ovalbumins, and ovomucoid, contain Gal1–4Gal (1), and all of their constituent N-glycans were found to possess Gal1–4Gal at the non-reducing termini of tri-, tetra-, and penta-antennary structures (2). No sialylation is found on the branch that contains the Gal1–4Gal sequence. Because biosynthesis and glycosyl modification of major egg white glycoproteins of chicken are carried out in tubular gland cells of oviduct (3), the most likely site of fabricating Gal1–4Gal linkages on the PEW glycoproteins would be in the corresponding cells of pigeon oviduct.

Gal1–4Gal in glycoproteins is, however, uncommon in nature. Mammals (e.g. human, pig, rat, mouse, and hamster) express Gal1–4Gal mostly on glycolipids (4–8), as in P₁ (Gal1–4Gal1–4GlcNAc1–3Gal1–4Glc1–1Cer) and P^k (Gal1–4Gal1–4Glc1–1Cer) antigens, but rarely on glycoproteins. An exception in mammals is the case of glycoproteins with Gal1–4Gal in hydatid cyst fluid and membrane infected by tapeworm Echnococcus granulosus (9–11). In animals other than birds and mammals, the only recorded presence of Gal1–4Gal was in O-glycosides such as those found in an insect cell line from Mamestra brassicae (12) or egg jelly mucins from amphibians, such as Xenopus laevis (13), Rana clamitans (14), and Ambystoma mexicanum (15).

The presence of Gal1–4Gal or substances similar to P₁ antigen on glycoproteins is limited even among other birds that have been studied. For example, P₁ antigenic activities is absent in the blood of chicken, gander, turkey, quail, duck, and pheasant (16, 17), and no -galactoside is found in the glycans of ovomucoid from chicken, quail, and duck (18–22). On the other hand, P₁ antigenic activities have been found in the blood and/or eggs of pigeon, turtle dove (Streptopelia resoria), budgerigar, and cockatiel (16, 23, 24). Salivary gland mucin glycoproteins of Chinese swiftlets (genus Collocalia) contain O-linked glycans with Gal1–4Gal sequence (25).

The biological significance of Gal1–4Gal in pigeon and some other avian glycoproteins is still unknown. Whether Gal1–4Gal is located specifically on PEW glycoproteins or glycoproteins of other pigeon organs is still unsettled. François-Gérard and co-workers (16, 17) reported the presence of P₁ antigenic activity in pigeon blood. However, they did not indicate whether the antigenicity was located on glycolipids or glycoproteins and whether the P₁ antigens were produced by pigeon themselves or derived from foreign bodies. To understand further the characteristics of species-specific oligosaccharides, we investigated the presence of glycoproteins with Gal1–4Gal in pigeon serum, lymphocyte, and liver. Here we report that one of the major glycoproteins containing Gal1–4Gal in serum is pigeon IgG, which possesses complex-type N-glycans containing Gal1–4Gal as well as high mannose-type oligosaccharides. The high mannose-type N-glycans are exclusively found on the CH3 domain, and the site specificity is probably a characteristic in avian IgGs. Unlike IgGs in other animals, the complex-type glycans were mainly tri- as well as bi-antennary structures. Moreover, N-glycans containing both Gal1–4Gal1–4Gal1–4GlcNAc and Gal1–4Gal1–4GlcNAc branches were detected by MS/MS analysis, which were not found in PEW glycoproteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Adult (female) pigeons were from a local racing pigeon breeder in Virginia. Pigeon livers were obtained from NASCO (Fort Atkinson, WI). Human serum, alkaline phosphatase-conjugated goat anti-mouse IgM, tributylphosphine, and 4-vinylpyridine were purchased from Sigma. Glycoamidase F² (GAF) was from Prozyme Inc. (San Leandro, CA). Trypsin (sequencing grade) was from Worthington. Alkaline phosphatase-conjugated GS-I lectin was purchased from EY Laboratories, Inc (San Mateo, CA). Anti-P₁ mAb (mouse IgM) was from Gamma Biologicals, Inc. (Houston, TX). 5-Bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium kit for alkaline phosphatase assay was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Ficoll-Paque^TM Plus was from Amersham Biosciences. Dulbecco's phosphate-buffered saline (10x) was purchased from Invitrogen. Chicken IgG and sera from chicken and bovine were from Pel-Freez Biologicals (Rogers, AR). BCA^TM Protein Assay reagents were from Pierce. Polyvinylidene difluoride (PVDF) membranes for blotting, ultrafiltration membrane (YM-10), and Centricon YM-10 were from Millipore (Bedford, MA). Superdex 200 columns for gel filtration (HR 10/30, 1.0 x 30 cm and HiLoad 26/60, 2.6 x 60 cm) and DEAE-Sepharose Fast Flow column (HiTrap, 1 ml) were from Amersham Biosciences. Shim-Pack CLC-ODS column (6.0 x 150 mm) was from Shimadzu Co. (Kyoto, Japan). TSKgel DEAE-5PW column (7.5 x 75 mm) and TSKgel Amido-80 column (4.6 x 250 mm) were from TosoHaas (Montgomeryville, PA). Carbograph tubes (25 mg) were from Alltech Associates.

Buffers and Standard Procedures—Tris-buffered saline contains 50 mM Tris·HCl (pH 7.4) and 150 mM NaCl. TBST contains 0.1% Tween 20 in Tris-buffered saline. Guanidine·HCl (8 M) was made in 0.2 M Tris·HCl (pH 8.0). Procedures for SDS-PAGE, Griffonia simplicifolia-I lectin blotting, and N-terminal sequence analyses have been described previously (1). Sequence identity search was performed using NCBI data bases. Protein concentrations were measured by the BCA assay (26) using bovine serum albumin (BSA) as a standard.

Preparation of Extracts from Pigeon Tissues and Cells—Blood (5 ml/pigeon) drawn from six pigeons were collected in blood collection tubes (13 x 75 mm) containing 0.057 ml of 15% K₃EDTA solution (BD Biosciences). The lymphocytes were isolated with Ficoll-Paque^TM Plus and washed with Dulbecco's phosphate-buffered saline twice. Pellets of lymphocytes and recovered plasma were stored at–20 °C until use. The lymphocytes and a portion of pigeon liver were homogenized in 100 mM sodium cacodylate (pH 7.0) containing 1% Triton X-100 at 4 °C. The supernatants separated by centrifugation were used as extracts.

Isolation of Major Glycoproteins from Pigeon Plasma—Diluted pigeon plasma (6.2 mg of protein/ml) was centrifuged to remove insoluble materials. Supernatant was filtered through a 0.45-µm membrane and injected onto a Superdex 200 column (maximum 0.2 ml for an HR 10/30 column, and 1 ml for a HiLoad 26/60 column) equilibrated with 50 mM sodium phosphate (pH 7.0) containing 150 mM NaCl. The flow rates were 0.25 ml/min for the HR 10/30 column and 2 ml/min for the HiLoad 26/60 column. Proteins in the eluate were monitored by A_280nm, and individual peaks were collected manually. GS-I-positive fractions were concentrated with ultrafiltration (YM-10 membrane) and desalted by washing with 10 mM Tris·HCl (pH 8.0) for further purification by ion exchange chromatography. A column of DEAE-Sepharose Fast Flow (HiTrap, 1 ml) was washed with 1 M NaCl in 10 mM Tris·HCl (pH 8.0) and equilibrated with 10 mM Tris·HCl (pH 8.0). After the sample injection, the column was washed with 10 mM Tris·HCl (pH 8.0) for 30 min (flow rate, 1 ml/min), and then the concentration of NaCl in the elution was linearly increased up to 0.4 M within 80 min (flow rate, 0.5 ml/min). The major peak was collected and concentrated with a Centricon YM-10.

GAF Treatment of Glycoproteins—Glycoprotein samples (10 µg each) in 20 µl of 0.4% SDS and 100 mM 2-mercaptoethanol in 10 mM Tris·HCl (pH 8.0), were heat-denatured at 90 °C for 3 min. After the solutions were cooled to room temperature, 1% (v/v) Nonidet P-40, and 1 unit of GAF were added to the solution of glycoproteins. The reaction mixtures were incubated at 37 °C for 16 h to complete de-N-glycosylation and then heated at 100 °C for 5 min to inactivate GAF.

Preparation of Glycopeptides for Peptide Sequencing—To reduce the existing disulfide bonds and to block random reformations of intramolecular disulfide bonds, 16 µmol of tributylphosphine and 0.16 mmol of 4-vinylpyridine were added to 1 mg of pigeon IgG in 0.1 M Tris·HCl (pH 8.5) and incubated at room temperature for 2 h under nitrogen. The reaction mixture was dialyzed against water and lyophilized. One-third of the reduced and alkylated pigeon IgG was suspended with 40 µlof50 mM NH₄HCO₃ (pH 8.4) and incubated with 12.5 µg of trypsin (sequencing grade) at 37 °C, overnight. A portion of the supernatant was further treated with GAF (0.5 units/10 µl) at 37 °C, overnight. The peptide mixtures, before and after the GAF treatment, were analyzed with reversed phase-HPLC on a Shim-pack CLC-ODS column. The mobile phases were solute A, 0.05% trifluoroacetic acid, and solute B, 90% CH₃CN in the 0.05% trifluoroacetic acid. Elution (1 ml/min) was conducted by a linear gradient of 0–50% of solute B in solute A developed over 100 min. Each peak monitored by A_210nm was collected and kept at 4 °C. A portion of the isolated glycopeptides was treated with GAF, and the released oligosaccharides and the deglycosylated peptide were separated with HPLC.

Preparation of Oligosaccharides for Structural Analyses—Pigeon IgG (1 mg) was reduced with 180 µl of 60 mM dithiothreitol in 8 M guanidine·HCl at room temperature for 1 h. For alkylation of thiols, 240 µl of 0.18 M iodoacetamide in 8 M guanidine·HCl was added, and the mixture was incubated at room temperature for 30 min in the dark. The reaction mixture was dialyzed against water and lyophilized. The alkylated pigeon IgG was suspended with 200 µl of 50 mM NH₄HCO₃ (pH 8.4), digested with 10 µg of trypsin at 37 °C for 4 h, and followed by addition of 10 µg of chymotrypsin (37 °C overnight). After inactivating the enzymes at 100 °C for 10 min, the digest was lyophilized. Oligosaccharides were released with GAF treatment in 50 mM NH₄HCO₃ (pH 7.8) at 37 °C overnight. After inactivating GAF at 100 °C for 5 min, the digest was lyophilized. To remove inorganic cations and peptides, the mixture was loaded onto 1 ml of Dowex 50W x 2 (H⁺ form, 50–100 mesh, Sigma) packed in a 1-ml syringe. The column was washed with 5 ml of water, and the collected effluent was lyophilized. The sample was dissolved with 200 µl of water, loaded onto a Carbograph tube (25 mg, Alltech), washed with 1 ml of water, and then eluted with 500 µl of 25% CH₃CN containing 0.05% trifluoroacetic acid.

Exo-glycosidase Digestion—The released N-glycan mixtures were digested sequentially with 50 milliunits of neuraminidase from Arthrobacter ureafaciens (Roche Applied Science) in 25 µlof50mM sodium acetate buffer (pH 5.0), 0.5 units of -galactosidase (from green coffee bean, Calbiochem) in 100 µl of 50 mM citrate-phosphate buffer (pH 6.0), followed by 5 milliunits of 1–4 galactosidase (from Streptococcus pneumoniae, Calbiochem) in 50 µl of 50 mM sodium acetate buffer (pH 6.0). All digestions were carried out at 37 °C for 24 h. Each enzyme digestion was desalted by passing through a mixed bed ion-exchange column packed with a mixture of 1 ml each of Dowex 50W-X8 (50–100 mesh, H⁺ form) and AG 3-X4 (100–200 mesh, free base form, Bio-Rad) resins, prior to taking an aliquot for permethylation and MS analysis. PA-glycans were desalted instead by passing through a Sep-Pak C18 cartridge (Waters), washed with water, and then eluted with 50% methanol in water.

Chemical Derivatization and GC-MS Linkage Analysis—N-Glycan and PA-derivatized N-glycan samples were permethylated using the NaOH/dimethyl sulfoxide slurry method as described by Dell et al. (27). For GC-MS linkage analysis, partially methylated alditol acetates were prepared from permethylated derivatives by hydrolysis (2 M trifluoroacetic acid, 121 °C, 2 h), reduction (10 mg/ml NaBH₄, 25 °C, 2 h), and acetylation (acetic anhydride, 100 °C, 1 h). GC-MS was carried out using a Hewlett-Packard Gas Chromatograph 6890 connected to a Hewlett-Packard 5973 Mass Selective Detector. Sample was dissolved in hexane prior to splitless injection into a HP-5MS fused silica capillary column (30 m x 0.25 mm inner diameter). The column head pressure was maintained at around 8.2 pounds/square inch to give a constant flow rate of 1 ml/min using helium as carrier gas. Initial oven temperature was held at 60 °C for 1 min and increased to 90 °C in 1 min and then to 290 °C in 25 min.

MS Analysis for Glycans and Glycomics—For MALDI-TOF MS glycan profiling, the permethylated derivatives in acetonitrile were mixed 1:1 with 2,5-dihydroxybenzoic acid matrix (10 mg/ml in acetonitrile), spotted on the target plate, air-dried, and recrystallized on-plate with ethanol whenever necessary. Data acquisition was performed manually on a benchtop MALDI LR system (Micromass) operated in the reflectron mode. For 2,5-dihydroxybenzoic acid matrix, the coarse laser energy control was set at high and finely adjusted using the % slider according to sample amount and spectra quality. Laser shots (5 Hz, 10 shots/spectrum) were accumulated until a satisfactory signal to noise ratio was achieved when combined and smoothed. Glycan mass profiling was also performed on a dedicated Q-TOF Ultima MALDI instrument (Micromass) in which case the permethylated samples in acetonitrile were mixed 1:1 with -cyano-4-hydroxycinnamic acid matrix (in acetonitrile, 0.1% trifluoroacetic acid, 99:1, v/v) for spotting onto the target plate. The nitrogen UV laser (337 nm wavelength) was operated at a repetition rate of 10 Hz under full power (300 µJ/pulse). MS survey data were manually acquired, and the decision to switch over to CID MS/MS acquisition mode for a particular parent ion was made on-the-fly upon examination of the summed spectra. Argon was used as the collision gas with a collision energy manually adjusted (between 50 and 200 V) to achieve optimum degree of fragmentation for the parent ions under investigation.

Off-line nanoelectrospray (nano-ESI) using the borosilicate metal-coated glass capillary option was performed on a Q-TOF Ultima API instrument (Micromass) equipped with a nanoflow source, mainly for CID MS/MS analysis of permethylated glycans samples. The capillary voltage was set at about 1.0–1.2 kV. Either protonated and/or sodiated doubly or triply charged parent ions may be selected for MS/MS. A collision energy setting of 20–40 eV was usually sufficient for doubly protonated species, but 60–80 eV was needed for singly and doubly sodiated parent ions. Permethylated samples were dissolved in 50% acetonitrile, 0.2% formic acid solvent and a 2-µl aliquot was typically loaded into the capillary for nano-ESI analysis.

Preparation and Isolation of PA-derivatized Oligosaccharides—Lyophilized free oligosaccharide fractions (from 2 mg of pigeon IgG) were dissolved in 40 µl of 2-aminopyridine solution (1 g of 2-aminopyridine in 580 µl of concentrated HCl (pH 6.8)) and heated at 90 °C for 15 min with heating block. Freshly prepared NaCNBH₃ solution (7 mg/4 µl water) was added into the reaction mixture and then heated at 90 °C for 1 h. PA-oligosaccharides were fractionated by gel filtration on a Sephadex G-15 column (1.0 x 40 cm, in 10 mM NH₄HCO₃), monitored by a fluorescence (excitation, 300 nm; emission, 360 nm), and lyophilized. The mixture of PA-oligosaccharides was first separated by HPLC with a TSK gel DEAE-5PW column (7.5 x 75 mm) as described previously (28), and the separated neutral, mono-sialyl, and di-sialyl fractions (monitored by fluorescence, Ex, 320 nm; Em, 400 nm) were collected separately and lyophilized. These fractions were individually dissolved in water and separated on a Shim-Pack CLC-ODS column (6.0 x 150 mm) as described previously (1). Elution was performed at a flow rate of 1.0 ml/min at 55 °C using eluent A (0.005% trifluoroacetic acid) and eluent B (0.5% 1-butanol in eluent A). The column was equilibrated with a mixture of eluents A/B = 90:10 (v/v), and after injection of a sample, the composition of the eluents was changed linearly to A/B = 30:70 in 120 min. Each peak was collected, lyophilized, and analyzed with MALDI-TOF-MS. For determination of N-glycan structures by two-dimensional mapping with HPLC, the isolated PA-oligosaccharides were analyzed using CLC-ODS and TSK gel amide-80 columns (4.6 x 250 mm) as described previously (29). Elution position of each of PA-oligosaccharides on CLC-ODS and amide-80 columns was expressed as glucose units (GU), based on the elution position of isomalto-oligosaccharide series. Reference PA-oligosaccharides from asialo-human IgG, bovine RNase B, and chicken serum IgG were prepared by the same method. Structures of the reference compounds are shown in Fig. 9. PA-derivatized N-glycans B-1, C-1, and D (Fig. 9) were obtained from -fucosidase and N-acetyl--D-hexosaminidase-digested PA-oligosaccharides from human IgG. N-Glycans B-2 and C-2 were prepared from B-1 and C-1, respectively, with -galactosidase digestion.

View larger version (26K): [in this window] [in a new window]   FIG. 9. Structures of reference oligosaccharides isolated from human IgG, bovine RNase B, and chicken serum IgG. PA-derivatized these N-glycans were utilized for HPLC analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Detection of -Galactoside on Pigeon Glycoproteins by GS-I Lectin and Anti-P₁ mAb

View larger version (36K): [in this window] [in a new window]   FIG. 1. Detection of -galactosyl residue on pigeon glycoproteins by lectin and immunoblotting. A, expression of Gal1–4Gal in pigeon. Proteins (5 µg each) from pigeon serum, liver, lymphocyte, and egg whites were heat-denatured with sample buffer containing 3% SDS and 5% 2-mercaptoethanol and separated by SDS-PAGE (12.5%). After electrophoresis, the proteins were transferred to PVDF membranes and stained with CBB, GS-I lectin, or anti-P1 mAb. B, comparison of the presence of -galactosyl residue on pigeon, chicken, bovine, and human sera glycoproteins from by GS-I lectin blotting. Ten µg of proteins from sera were separated by SDS-PAGE, transferred to a PVDF membrane and stained with CBB or GS-I.

Isolation of Pigeon Plasma Glycoproteins
Pigeon plasma proteins (600 µg total) were separated by gel filtration using a Superdex 200 HR 10/30, as shown in Fig. 2A. As molecular size markers, chicken IgG (170 kDa) and BSA (67 kDa) were eluted in separate runs, and their positions were marked. Each of the peaks obtained from the Superdex 200 was collected, and 50 µl of each fraction was analyzed by SDS-PAGE and GS-I lectin blotting under the reducing conditions (Fig. 2, B and C). Proteins in fractions 2–8 were positively stained by GS-I, whereas other fractions (fractions 1 and 9–16) were negative to GS-I. The fraction 13 was the largest peak detected by A_280nm, but no protein/peptide bands were visualized with CBB or GS-I staining. One of the glycoproteins in the fraction 6, which was eluted at the same elution position as chicken IgG, showed the strongest intensity by the GS-I staining. In contrast, major proteins in fraction 8, where BSA is expected to elute, were stained with CBB strongly but not at all with GS-I. The similar elution profile was detected when 6 mg of pigeon plasma proteins was separated using a semi-preparative Superdex 200 column (2.6 x 60 cm) (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Isolation of -galactosylated glycoproteins from pigeon plasma. A, separation of pigeon plasma proteins by Superdex 200 HR 10/30. Six hundred µg of pigeon plasma proteins were loaded onto a Superdex 200 HR 10/30 column (1.0 x 30 cm) and eluted by 50 mM sodium phosphate (pH 7.0) containing 150 mM NaCl. Positions of reference molecules (chicken IgG and BSA) are shown. Individual peaks from the column were collected and analyzed on SDS-PAGE, using CBB staining (B) and GS-I lectin blotting (C). D, purification of the major glycoprotein from pigeon plasma with DEAE-Sepharose. Desalted fraction (fr.) 6 from the Superdex 200 column was applied to a DEAE-Sepharose column (1 ml) and eluted with a linear gradient of NaCl in 10 mM Tris·HCl (pH 8.0). The major peak (fr. 3) was collected and analyzed by SDS-PAGE (inset).

Identification of the Pigeon Serum Glycoprotein by Partial Peptide Sequencing
N-terminal amino acid sequences of the light (L) and heavy (H) chains were homologous to the corresponding variable regions of immunoglobulins of chicken, duck, and human (Table I). Because avian IgG, IgA, and IgM share the same N-terminal variable domains (35), internal peptide sequencing on the Fc region would be required for the determination of the immunoglobulin classes (36–38). The predominant serum immunoglobulin in birds is IgG, also known as IgY, because of its unique structure. It is functionally equivalent to mammalian IgG but has one additional constant region domain in its heavy (H) chains (Fig. 3) (33, 39). Throughout this paper, we refer to the predominant serum immunoglobulin in birds as serum IgG for convenience. The amino acid sequences of chicken and duck immunoglobulin chains (H chains) suggest that two potential N-linked glycosylation sites are indeed well conserved between chicken and duck, located in the Fc region (Fig. 3) (36, 40). In contrast, positions of potential N-glycosylation sites on Fc regions of avian IgA and IgM are different from those of IgG. Therefore, isolation and peptide sequencing of N-glycosyl peptides were performed in order to determine the immunoglobulin class based on sequence homology. The C18-HPLC elution profiles of tryptic peptides of pigeon IgG, before and after GAF treatment, indicated that "Peak A" shifted its position from 46.4 to 50.1 min after GAF treatment (Fig. S1). As shown in Table I, the glycopeptide sequence of the pooled peak A is homologous to that of a CH3 domain of chicken or duck IgG. The sequence alignment showed that conservation of the potential N-glycosylation sites is extended to pigeon, chicken, and duck but not to chicken IgM and IgA. Based on these sequence data, the isolated protein was identified as pigeon serum IgG. The CH3 domains of chicken and duck IgG resemble the CH2 domain of mammalian IgG (36, 39, 40). This appears to be the case for pigeon IgG also, because the peptide sequence containing the N-glycosylation site was similar to the corresponding site of the CH2 domains of human (Table I) and other mammalian IgG (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 3. Structural diagram of avian IgG. The domains of the light chains are termed VL (in the variable region) and CL (in constant region). Typical avian IgG has five domains in the heavy chain, termed VH (in variable region), CH1, CH2, CH3, and CH4 (in the constant region). Position of potential N-glycosylation sites (shown as hexagons) were based on chicken IgG H chain ( chain) (36). Numbering for chicken immunoglobulin chain is based on the deduced amino acid sequences from cDNA, starting from the first methionine in the leader region (36, 38, 67). Variable regions do not always have N-glycosylation. Location of inter-chain disulfide bonds were predicted in analogy to those of human IgE (36, 68) and indicated as dotted boldface lines.

Release of -Galactoside-containing Oligosaccharides from Pigeon IgG with GAF

MS and MS/MS Analysis of N-Glycans from Pigeon IgG
The N-glycans released by GAF from pigeon IgG were first profiled by MALDI-TOF MS analysis of the permethylated derivatives. Substantial heterogeneity was evident (Fig. 4A), but the glycans can be conveniently categorized into the following: (i) high mannose-type, (ii) disialylated biantennary complex-type, and (iii) -galactosylated complex-type structures.

View larger version (30K): [in this window] [in a new window]   FIG. 4. MALDI-TOF MS of the permethylated N-glycans from pigeon IgG before (A) and after (B) desialylation by neuraminidase. The labeled m/z values correspond to the most abundant isotope peak detected as [M + Na]+ molecular ions, except for m/z 2097 and 2301, which are oxonium-type fragment ions from Hex9,10HexNAc2 (m/z 2397 and 2601), the two most abundant components. The major non-sialylated complex type structures (resistant to neuraminidase) could be assigned as Hex4–8HexNAc4CF (m/z 3144, 3349, 3553, 3757, and 3962); Hex4–8HexNAc4C (m/z 2969, 3174, 3379, 3583, and 3788); and Hex2–5HexNAc3CF (m/z 2490, 2694, 2898, and 3103), where C and CF denote trimannosyl core and fucosylated trimannosyl core, respectively. The corresponding profile of the N-glycans from the isolated glycopeptides on CH3 domain (C) was devoid of the complex-type structures. The mass region from m/z 2800–4000 was magnified to show that no signal was detected.

Disialylated Biantennary N-Glycans—The sialylated components were first identified by the m/z values of the molecular ion signals and subsequently confirmed by digestion with neuraminidase (Fig. 4B). A prominent desialylated component afforded a major molecular ion at m/z 2071 corresponding to a biantennary complex-type structure that was absent before desialylation (Fig. 4A). The biantennary branching pattern was established by MS/MS analysis (Fig. S3). In particular, the ion at m/z 1143 from MALDI-MS/MS (Fig. S3B) unambiguously defined the presence of two antennae extending from the trimannosyl core, whereas the ion pair at m/z 432 and 464 from nano-ESI-MS/MS (Fig. S3A) indicated an N-acetyllactosamine (LacNAc) unit and not a type 1 chain (Gal1–3GlcNAc) (27). Removal of two Hex units by 4-galactosidase from S. pneumoniae resulted in a shift of the corresponding molecular ion signal at m/z 2071 (Fig. 5A) to m/z 1661 (Fig. 5B) which confirmed this assignment. Finally, methylation analysis of the desialylated samples clearly demonstrated a disappearance of 6-linked Gal (data not shown) as compared with the GC-MS profile of the sample not treated by neuraminidase (Fig. S4). The molecular ion at m/z 2794 in Fig. 4A could thus be assigned as 2–6-disialylated biantennary complex-type N-glycan which shifted to give a molecular ion of m/z 2071 after neuraminidase digestion (Fig. 4B) and remained resistant to -galactosidase digestion (Fig. 5A). No other sialylated component of high abundance was apparent from the analysis of the total mixtures, although monosialylated counterparts of several other complex-type structures could be detected as minor peaks, the detailed analysis of which was made possible after HPLC fractionation (see below).

View larger version (22K): [in this window] [in a new window]   FIG. 5. MALDI-TOF MS of the permethylated N-glycans from pigeon IgG after sequential digestion with neuraminidase, -galactosidase (A), and 4-galactosidase (B). As demonstrated by MS/MS analysis (Figs. 6 and 7), most of the non-sialylated complex-type structures carried a bisecting GlcNAc. The triantennary Hex4–8HexNAc4CF (m/z 3144, 3349, 3553, 3757, and 3962 in Fig 4B) and Hex6–8HexNAc4C components (m/z 3379, 3583, and 3788 in Fig. 4B) therefore carried a maximum of three terminal -Gal and were trimmed to Hex2–5HexNAc4CF (m/z 2735, 2939, 3143, and 3348) and Hex3–5HexNAc4C (m/z 2765, 2969, and 3173), respectively, after -galactosidase digestion (A), corresponding to removal of two or three -Gal residues. Likewise the bisected biantennary Hex2–5HexNAc3CF (m/z 2490, 2694, 2898, and 3103 in Fig. 4B) carried a maximum of two terminal -Gal and could be trimmed to Hex1–3HexNAc3CF (m/z 2286, 2490, and 2694), corresponding to removal of one or two -Gal residues. The signal at m/z 2735 (Hex2HexNAc4CF) in A could also derive from incomplete digestion by -galactosidase, leaving an -Gal-capped antenna resistant to further -galactosidase digestion (B). The signal at m/z 1711 was contributed by the MALDI matrix used.

When the major molecular ion signals from samples not treated with the exoglycosidase (Fig. 4A) were subjected to MALDI CID MS/MS analysis, additional unique structural features were unveiled. Fig. 6 shows spectra of MS/MS analysis for the Hex_4–8HexNAc₄CF series, where CF denotes fucosylated trimannosyl core. Interpretation of the MS/MS data for the Hex_4–8HexNAc₄CF series was summarized in Fig. 7. As expected, the presence of Gal-Gal-GlcNAc terminal epitope was indicated by the sodiated b ion at m/z 690 which could be detected in all of the glycans analyzed. For the larger components, however, another b ion at m/z 894, corresponding to sodiated Hex₃HexNAc⁺, was also very prominent. In the case of Hex₈HexNAc₄CF, sequential loss of the Hex₂HexNAc or Hex₃HexNAc terminal units concomitant with emergence of free OH group(s) yielded the complementary set of y ions (e.g. at m/z 3292, 2420, 1548, 3088, and etc., see Fig. 7E). MS/MS data also indicated that the majority of the Hex₈HexNAc₄CF components is triantennary carrying a bisecting GlcNAc that is readily lost from the parent ion under the MS/MS conditions employed. Two ions at m/z 1548 (corresponds to the fucosylated trimannosyl core with a bisecting GlcNAc and three free OH groups resulting from the loss of all three antennary sequences, Fig. 7E, left) and m/z 1289 (corresponds to a further loss of the bisecting GlcNAc giving a total of four free OH groups, Fig. 7E, right), which were present in all Hex_4–8HexNAc₄CF series structures, supported the presence of bisecting GlcNAc. Thus, the structure of Hex₈HexNAc₄CF is most likely a triantennary bisected and fucosylated trimannosyl core, extended with one Hex-Hex-HexNAc (Hex₂HexNAc) and two Hex-Hex-Hex-HexNAc (Hex₃HexNAc) sequences (Fig. 7E).

View larger version (31K): [in this window] [in a new window]   FIG. 6. MALDI CID MS/MS of permethylated -galactosylated triantennary complex-type structures from pigeon IgG. A–E refer to Hex4–8-HexNAc4CF, as labeled on the figure. Assignment of the fragment ions are illustrated by diagrams in Fig. 7. Some fragment ions could not be labeled or assigned, because of complication arising from isomeric structures.

View larger version (35K): [in this window] [in a new window]   FIG. 7. Summary of the major -galactosylated triantennary complex-type structures as deduced from MS/MS sequencing data (Fig. 6). A, Hex4HexNAc4CF; B, Hex5HexNAc4CF; C, Hex6HexNAc4CF; D, Hex7HexNAc4CF; and E, Hex8HexNAc4CF. Full assignment is shown for Hex8HexNAc4CF (E) where the glycosidic oxygen (-O-) are shown to illustrate the sites of cleavage. The majority of the ions are either b or y ions. The b ions at m/z 690, and 894 showed that both Hex2HexNAc+ and Hex3HexNAc+ termini are present. A and B, the presence of incompletely galactosylated termini was also evident from m/z 282 (HexNAc+) and 486 (Hex1HexNAc+). A combination of multiple cleavages produced the complement of y ions unique to each structure. A facile loss of the bisecting GlcNAc was illustrated in E but indicated as further loss from the y ions with an arrow in other structures for simplicity (A–D). All structures from the Hex4–8HexNAc4CF series gave the common ions at m/z 1548, 1289, 838, 719, 648, and 474 which were shown only in E. Another common ion at m/z 648 (Fig. 6) is related to m/z 838 (Figs. 6 and 7E, left), representing a further loss of the branched terminal Hex (Man) residue. For Hex4,5HexNAc4CF, the more abundant isomer could be represented by the structures on the right in B and A, but the presence of the isomers shown on the left was evident from the additional ions detected. The location of the different antennae on either 3- or 6-arm of the trimannosyl core was not defined. Symbols used: triangle, Fuc; square, HexNAc; circle, Hex; the darker filled circle at the non-reducing termini is meant to represent -Gal capping although MS data cannot be used to distinguish it from other Hex.

The predominance of bisected complex-type structures was in harmony with the methylation analysis (Fig. S4). The presence of unsubstituted GlcNAc and a triply substituted (3,4,6-linked) Man supported this conclusion. Only 4-linked GlcNAc was found (indicative of the type 2 chain, Gal1–4GlcNAc), and the amount of 3-linked GlcNAc residue was insignificant. The Gal residue linked to GlcNAc was susceptible to 4-galactosidase. The linkage analysis data suggest that Hex₂HexNAc and Hex₃HexNAc terminal units may be Gal-4Gal-4GlcNAc and Gal-4Gal-4Gal-4GlcNAc, respectively, because no other residue, e.g. branched Gal residue, was found. The results of digestion using - and 4-galactosidases (Fig. 5) suggest that they are most likely to be Gal1–4Gal1–4GlcNAc1– and Gal1–4Gal1–4Gal1–4GlcNAc1–. These sequences were further demonstrated using fractionated oligosaccharides (see below).

Fractionation and Structural Analysis of PA-derivatized N-Glycans from Pigeon IgG
The presence of Gal₃GlcNAc antenna was unexpected and has resulted in greater heterogeneity, because each glycan as assigned above may consist of smaller amounts of isomers with different combinations of fully galactosylated to non-galactosylated antennae. This was found to be the case when the glycans from pigeon IgG were derivatized with 2-aminopyridine and fractionated first into neutral, monosialylated, and disialylated fractions with a DEAE column (Fig. 8A) and then further fractionated on an ODS column (Fig. 8B), followed by MS and MS/MS analyses of the collected major fractions (Tables II and III and Supplemental Material Figs. S1 and S2). Molar ratios of pigeon IgG N-glycans calculated from the peak areas of PA-oligosaccharides were neutral 64.8% (high mannose-type, 33.3%, others, 31.5%), monosialylated 16.0%, and disialylated 19.2%. N-Glycan structures of the three categories were determined as follows.

View larger version (28K): [in this window] [in a new window]   FIG. 8. Separation of PA-oligosaccharides from pigeon serum IgG by HPLC. A, total PA-oligosaccharides from pigeon IgG were separated into neutral, mono-, and disialyl oligosaccharides on the DEAE column. B, elution profiles of the neutral, mono-, and disialyl PA-oligosaccharides on the ODS column. Each peak was analyzed with mass spectrometry. Fraction numbers for N-glycans correspond to those in Tables II and III and Supplemental Material Tables S1 and S2.

PA-derivatized Disialylated Biantennary N-Glycans—The two disialylated N-glycans (ds-6 and ds-8) were analyzed by MALDI-MS before and after permethylation to define their molecular compositions and by MALDI MS/MS and HPLC for structural assignment (Table III). N-Glycan ds-8 clearly corresponded to the disialylated biantennary structures detected in the MS analysis of the total mixtures for which further MS/MS analysis of the desialylated structures (Fig. S3) have unambiguously established its biantennary structures. In addition, after -neuraminidase digestion, the elution positions of ds-8 on HPLC columns were indistinguishable from human IgG N-glycan D (Fig. S5B), confirming its structure as non-bisected biantennary complex-type without core fucosylation. MS and MS/MS analysis of N-glycans ds-6 (Fig. S5A and Table III) led to identification of a unique NeuAc-HexNAc-HexNAc antenna on ds-6 in which the Hex to HexNAc substitution constitutes the sole difference between ds-6 and ds-8. This was supported by the fact that the elution of ds-6 (GU_Amide, 6.9) on the amide-80 column was 0.3–0.4 GU_Amide earlier than that of ds-8 (GU_Amide, 7.2) (Fig. S5B), which is attributable to the difference in unit contribution values between Hex and HexNAc (29, 44). Likewise, the GU_Amide of both ds-6 and ds-8 was reduced by about 0.3–0.4 GU after desialylation, indicative of their 2–6 but not 2–3 sialylation (45, 46). When the desialylated ds-6 was further treated with N-acetyl--D-hexosaminidase, the elution positions on ODS and amide-80 columns (GU_ODS, 10.3; GU_Amide, 5.6) were indistinguishable from the reference N-glycan B-1 but different from C-1 (GU_ODS, 7.7; GU_Amide, 5.7). This suggests that desialylated ds-6 released two HexNAcs from the Man1–3 branch. Further digestion with -D-galactosidase resulted in a product eluting at the same position as N-glycan B-2 (GU_ODS, 9.5; GU_Amide, 4.7) but different from C-2 (GU_ODS, 7.1; GU_Amide, 4.6), thus further confirming that the NeuAc-HexNAc-HexNAc antenna of ds-6 is extending from the Man1–3 branch. NeuAc-HexNAc-HexNAc unit is most likely corresponding to NeuAc2–6GalNAc1–4GlcNAc, although no further evidence can be obtained at present because of lack of the sufficient sample quantity.

PA-derivatized -Galactosylated Neutral N-Glycans—The ODS-fractionated neutral complex-type structures (eluted over 20–70 min), identified by MS and MS/MS, are listed in Table S1. Each of the fractions (Fig. 8B) was first screened by MALDI-TOF MS, and the assigned composition was further confirmed by subsequent analysis of the permethylated derivatives. MALDI MS/MS was performed where the sample quantity permits, and the observed fragment ions collectively established the permutation of galactosylated antennae. In general, the fragmentation is similar to the underivatized samples (Fig. 7), except that each of the y fragment ions carrying the reducing terminal HexNAc would have a mass increment of 92 units due to the PA tag. For clarity, only the characteristic fragment ions essential for structural assignment are listed in Table S1. As illustrated in Fig. S6, the important features are as follows.

1. Core fucosylation was assigned if a prominent ion at m/z 566 was detected, corresponding to (OH)(Fuc)GlcNAc-PA. This is further supported by b ions resulting from cleavage at the chitobiose core.
   
2. Sodiated b ions at m/z 894, 690, and/or 486 identified the presence of Hex₃HexNAc-, Hex₂HexNAc-, and/or Hex₁HexNAc-antennae, respectively. This was corroborated by sequential loss of the antennary units from the parent ions, giving rise to a series of y ions. The presence of a bisecting GlcNAc was indicated by detection of a facile loss of terminal HexNAc from the parent and other y ions.
   
3. Multiple cleavages often resulted in a pair of characteristic b ions corresponding to the trimannosyl core, and the number of free OH groups contained can be used to infer the number of antennae attached originally.
   

Conclusions that can be drawn from more detailed MS/MS analysis of the individually purified components are in general consistent with the analyses of the total mixtures (Fig. 4A), except that ODS fractionation of the PA-derivatives separated isomeric series and also led to detection of a more complete range of minor components.

-Galactosidase digestion of Hex₇HexNAc₄CF-PA from n-37 resulted in a loss of three -Gal residues, and subsequent 4-galactosidase digestion converted it to HexNAc₄CF-PA (Fig. S7). Together with the MS/MS results (Table S1), the structure of n-37 is most likely (Gal1–4Gal1–4Gal1–4GlcNAc)-(Gal1–4Gal1–4GlcNAc)₂-NCF-PA, where NCF denotes fucosylated trimannosyl core with bisecting GlcNAc. Similarly, sequential /-galactosidase digestions of Hex₆HexNAc₄CF-PA from n-38 released two -Gal and four 4-Gal residues and thus implicated a likely structure of (Gal1–4Gal1–4GlcNAc)-(Gal1–4Gal1–4GlcNAc)₂-NCF-PA. These results indicated that for n-38, one of the Hex₂HexNAc antennae was not -Gal capped. The non-reducing termini of pigeon IgG N-glycans may therefore comprise Gal1–4Gal1–4GlcNAc, Gal1–4GlcNAc, and GlcNAc in sub-galactosylated structures.

PA-derivatized -Galactosylated Monosialylated N-Glycans—Although only a single disialylated biantennary structure was prominent, the presence of other minor sialylated structures was noted from MS analysis of the total mixtures (Fig. 4A). Indeed, after PA derivatization and fractionation on the ODS column, about 60 peaks could be detected for the monosialylated fraction. A select few of the monosialylated components were screened by MALDI-MS before and after permethylation to define their molecular compositions and, wherever possible, analyzed by MALDI MS/MS for structural assignment (Table S2). MS/MS analysis of the major monosialylated glycans indicated that all sialylated antennae are NeuAc-Hex-HexNAc with no additional Gal inserted (Fig. S8 and Table S2 and Fig. 10). However, the other antennae can be Hex₂HexNAc or Hex₃HexNAc, likely to be identical to the -galactosylated antenna found in the neutral glycans. It thus appears that the predominant bisected bi- and triantennary complex-type structures with core 6-fucosylation are highly heterogeneous due primarily to a combination of different capping moieties on the Gal1–4GlcNAc antennae, i.e. Gal1–4–, Gal1–4Gal1–, and NeuAc2–6–. In the case of Gal1–4Gal1–4Gal1–4GlcNAc termini, an additional -Gal may be added to the LacNAc unit without the concomitant -Gal capping. Although we have not analyzed all of the isomeric structures resolved by HPLC, the general conclusion may apply to the overall glycosylation heterogeneity of pigeon serum IgG.

View larger version (35K): [in this window] [in a new window]   FIG. 10. Summary of the major monosialylated structures as deduced from MS/MS sequencing. A, NeuAc1Hex3HexNAc3CF-PA from ms-42; B, NeuAc1Hex4HexNAc3CF-PA from ms-40; C, NeuAc1Hex5HexNAc4CF-PA from ms-50; and D, NeuAc1Hex6HexNAc4CF-PA from ms-48. In addition to the key fragment ions illustrated in Fig. S6, key y ions resulting from sequential loss of one or more of the antennae were also illustrated. As in Fig. 7, the facile loss of the bisecting GlcNAc is indicated by an arrow from the primary cleavage ions. The location of the different antennae on either 3- or 6-arm of the trimannosyl core was not defined. Symbols used are as in Fig. 7 and S6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Moreover, analysis of oligosaccharide structures of pigeon IgG unveiled several unique glycosylation properties not found in other avian IgGs or in mammalian IgGs. First, little or no -galactosylated oligosaccharides have been detected in normal serum IgG of non-primate mammals such as dog, cow, sheep, horse, rat, mouse, rabbit, and cat (42, 50), even though these animals have functional -1,3-galactosyltransferases (-1,3-GalT). A well conserved N-glycosylation site in mammalian IgG is located at Asn-297 (Eu numbering) on CH2 domains (51), and oligosaccharide chains can be seen occluding the cavity at the center of the Fc (52). The N-glycosylation at Asn-297 with biantennary complex-type oligosaccharides is indispensable, because it influences mammalian IgG in its thermal stability, interaction with Fc receptors, association with C1q, and induction of antigen-dependent cellular cytotoxicity (53–55). It is presumed that N-glycans in the cavity of Fc region cannot possess Gal1–3Gal termini, due to steric hindrance (56). If this is true, then the highly -galactosylated features of pigeon serum IgG require a different explanation. The rationalization came from analysis of oligosaccharides in the pigeon IgG-CH3 domain that is structurally equivalent to the mammalian IgG-CH2 domain (36, 39). The result indicated that no complex-type N-glycans were located in pigeon IgG-CH3 glycopeptides (Fig. 4C).

Second, about one-third of the total N-glycans from pigeon serum IgG was high mannose-type oligosaccharides, whereas normal mammalian serum IgG contain exclusively biantennary complex-type N-glycans (42). It should be pointed out that 61.7% of the high mannose-type glycans in pigeon IgG are mono-glucosylated as found in chicken (serum, egg yolk) (41) and quail (egg yolk) IgGs (43). Specifically, we demonstrated that pigeon IgG-CH3 glycopeptides contain only high mannose-type oligosaccharides. Because the cognate site specificity was also found in chicken serum IgG,³ this site-specific N-glycosylation pattern might have resulted from the steric hindrance imposed by the unique conformational structures of avian IgG. The steric hindrance on avian IgG-CH3 may be more severe than that on mammalian IgG-CH2 and allow only limited process on the N-glycans.

Because N-glycans located on CH3 domain are exclusively high mannose-type, complex-type N-glycans were consequently located at other glycosylation sites on avian IgG. Interestingly, structural analysis of N-glycans revealed that the complex-type N-glycans in IgG were very different between pigeon and chicken. This might be explained that the sites for complex-type N-glycans on these avian IgG are more exposed and accessible by "processing" enzymes, which may form more branched, longer N-glycans. The presence of triantennary N-glycans is unique in pigeon IgG, as it has not been seen in chicken and quail IgGs (41–43).

Third, we found a novel N-glycan structure containing Gal1–4Gal1–4Gal1–4GlcNAc branches on pigeon IgG. The presence of the structures could not be shown by lectin and immunostainings, because of the coexistence of Gal1–4Gal1–4GlcNAc, but MALDI CID MS/MS analysis identified these two branches. The result was surprising, because N-glycans of major PEW glycoproteins (2) as well as known N-glycan structures of chicken (41) and quail egg yolk IgG (43) do not contain Gal1–4Gal sequence at all. The absence of Gal1–4Gal on PEW glycoproteins may be because the putative -1,4-galactosyltransferase (Gal:-1,4-GalT) responsible for the synthesis of Gal1–4Gal is inactive in pigeon oviduct but is active in IgG-producing